9GKS
Crystal structure of artificial enzyme LmrR_pAF variant RMH in crystal form 2
Summary for 9GKS
| Entry DOI | 10.2210/pdb9gks/pdb |
| Descriptor | Transcriptional regulator, PadR-like family (2 entities in total) |
| Functional Keywords | artificial enzyme, unnatural amino acid, 4-aminophenylalanine, directed evolution, dna binding protein |
| Biological source | Lactococcus cremoris subsp. cremoris MG1363 |
| Total number of polymer chains | 2 |
| Total formula weight | 30326.35 |
| Authors | Thunnissen, A.M.W.H.,Leveson-Gower, R.B.,Rozeboom, H.J.,Roelfes, G. (deposition date: 2024-08-26, release date: 2025-02-26) |
| Primary citation | Leveson-Gower, R.B.,Tiessler-Sala, L.,Rozeboom, H.J.,Thunnissen, A.W.H.,Marechal, J.D.,Roelfes, G. Evolutionary Specialization of a Promiscuous Designer Enzyme. Acs Catalysis, 15:1544-1552, 2025 Cited by PubMed Abstract: The evolution of a promiscuous enzyme for its various activities often results in catalytically specialized variants. This is an important natural mechanism to ensure the proper functioning of natural metabolic networks. It also acts as both a curse and blessing for enzyme engineers, where enzymes that have undergone directed evolution may exhibit exquisite selectivity at the expense of a diminished overall catalytic repertoire. We previously performed two independent directed evolution campaigns on a promiscuous designer enzyme that leverages the unique properties of a noncanonical amino acid (ncAA) -aminophenylalanine (pAF) as catalytic residue, resulting in two evolved variants which are both catalytically specialized. Here, we combine mutagenesis, crystallography, and computation to reveal the molecular basis of the specialization phenomenon. In one evolved variant, an unexpected change in quaternary structure biases substrate dynamics to promote enantioselective catalysis, while the other demonstrates synergistic cooperation between natural side chains and the pAF residue to form semisynthetic catalytic machinery. PubMed: 39944761DOI: 10.1021/acscatal.4c06409 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (2.24 Å) |
Structure validation
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